The present invention relates to methods for producing embryos and regenerating plants from cultured microspores.
Doubled haploid plants can be generated from hapioid plants or cells in which the chromosome number is doubled to the normal somatic number(2n) by duplication. Methods for production of haploid and doubled haploid plants have application to plant breeding and transgenic plant production.
The induction of microspore division leading to embryogenesis has application to large-scale haploid and doubled haploid plant production. Methods are known which induce formation of embryo-Like structures from microspores in which genotypes within a species can be induced to produce large numbers of haploids. However, the regeneration of green plants from such embryos has been problematic using known methodology. Genotypes of some monocotyledonous species such as cereals produce albino plants from embryo-like structures. Conventional isolated microspore culture techniques have not led to consistent embryo regeneration, and disadvantageously result in the production of a high proportion of albino plants.
U.S. Pat. No. 4,840,906 (Hunter) teaches a method of plant regeneration from barley microspores incubated at 25xc2x0 C. for 28 days in a sugar-containing culture medium following 28 days of cold pre-treatment. The method disclosed results in wide variation in the rate of green plantlet formation from microspores, ranging anywhere from about 30 to about 200 green plantlets formed per 100 anthers cultured. There is clearly a need for an improved method that consistently produces high yields of green plantlets from isolated microspores.
U.S. Pat. No. 5,445,961 (Genovesi et al.) discloses a method for embryogenesis of microspores using a pretreatment of sugar alcohol and cold (about 10xc2x0 C.) which also requires colchicine, a chromosome doubling agent. This pretreatment is followed by microspore isolation and growth on culture medium. From 2 to 115 embryoids were produced for every 104 microspores incubated according to this method. Transformation of microspores after treatment with a chromosome doubling agent would be less likely to result in homozygous transformants than if transformation were to occur prior to chromosome doubling. However, no transgenic transformation of microspores is disclosed in this document.
The haploid single-celled microspore is an attractive target for mutation, selection, and transformation. When transformation is performed at the G1 phase of the nuclear cycle, genetically homozygous plants are produced which include transgenes introduced prior to division. Yao et al. (Genome 1997;40:570-581) treated highly inducible barley microspores of the genotype Igri with mannitol, followed by biolistic bombardment to transform the microspores. However, the regenerated plants were largely heterozygous for the transgene.
Jxc3xa4hne et al. (Theor Appl. Genet. 1994;89:525-533) describes a method of using barley isolated microspores for particle bombardment. From cold pretreated spikes, microspores were quickly isolated and maintained at about 24C for 1 hour prior to bombardment with a transgene. An average of 82 green plants per 105 microspores were produced according to the disclosed method, and only one transgenic plant formed for every 2.8xc3x97106 microspores.
Methodological conditions which contribute to successful methodology for embryogenesis, and plant regeneration include donor plant growth, pre-treatment, culture medium components, such as sugar type, nitrogen source and balance, hormones, and medium density and osmolality. However, a method resulting in a high rate of embryogenesis, and successful production of high numbers of green plants has not been disclosed. A methodology is required which employs a optimal and synergistic combination of the above-noted conditions, resulting in a high production rate of green plants from microspores. Such an improved method would be labor saving, and of higher efficiency than conventional methodology, thereby reducing production costs.
Arabinogalactan proteins (AGP) are plant proteoglycans present in a diverse number of plant tissues which may have regulatory functions in plant cell reproduction processes. Previous reports have illustrated positive effects of arabinogalactan proteins on somatic embryogenesis in Picea abies(Norway Spruce) and in Daucus carota L. (see, for example Egertsdoner et al. Physiol Plant 1995;93:334-45; and Kreuger et al. Planta 1993;189:243-248). Kawaguchi et al. (Plant Journal, 1996;9(6):777-785) describe a tetrasaccharide similar in structure to arabinogalactan protein, which accumulates in rice anthers in a stage-specific manner. The effects of AGP on embryogenesis and plant regeneration from microspores has not been evaluated.
European Patent Publication No. 0 455 597 A1 (Sandoz Ltd) discloses a method for stimulating growth of Daucus carota L cells plants in in vitro culture. This publication describes the stimulation of growth by adding AGP to culture medium at a level of from 0.01 to 100 mg/L. However, AGP was not used or assessed for efficacy in inducing embryogenesis of microspores. Additionally, no transformation methodology or staging of cells in culture is taught.
There is a need for a method of embryogenesis which results in the production of green plants with a high success rate.
There is also a need for an effective method of plant transformation from embryogenic induction of microspores, which provides an effective pre-treatment to synchronize microspores and holds them at the G1 phase of the cell cycle prior to transformation, thereby increasing the production of homozygous transformants.
It is an object of the invention to overcome disadvantages of the prior art. The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
The present invention relates to methods for producing embryos and regenerating plants from cultured microspores.
According to this invention, there is provided a method of embryo production comprising the steps of (a) harvesting a microspore-containing plant segment from a donor plant; (b) incubating the segment under pretreatment conditions to maintain a substantial portion of microspores at a uninucleate cell cycle G1 phase; (c) isolating microspores from the segment; and (d) incubating said isolated microspores in an induction medium comprising arabinogalactan protein to induce embryogenesis, thereby producing embryos.
Further, the invention provides a method of plant regeneration from micro spores comprising the steps of (a) harvesting a micro spore-containing plant segment from a donor plant; (b) incubating the segment under pre-treatment conditions to maintain a substantial portion of microspore uninucleate cell cycle G1 phase; (c) isolating microspores from the segment; (d) incubating the isolated microspores in an induction medium comprising an auxin to induce the production of embryos; (e) incubating the embryos in a differentiation medium to produce differentiated embryos; and (f) regenerating plants from the differentiated embryos.
The present invention is also directed to the production of an embryo that is prepared by the above method, and to a plant produced from this embryo.
This invention pertains to a method of introducing a gene of interest into a microspore comprising introducing a genetic construct comprising the gene of interest into the microspore following the steps of pretreatment and isolation. Methods for introducing the genetic construct into the microspore comprise particle bombardment or Agrobacterium mediated transformation. Furthermore, the present invention is directed to a transgenic microspore prepared by this method, and to a transgenic embryo and transgenic plant produced from this transgenic microspore and transgenic embryo, respectively.
Advantageously, the method is efficient and labor saving as compared with known microspore culture and anther culture procedures. The higher success rate for green plant production according to the inventive method will result in cost savings. A further benefit of the inventive methodology is a reduction in the number of albino plants produced.
Advantageously, the inventive method is effective across genotypes, illustrating the absence of a strong genotype effect. The synergistic combination of factors result in a versatile method which may be applied to many types of plants.
Advantageously, the removal of the anther wall during microspore isolation allows for better nutrient availability to microspores during induction, differentiation and regeneration steps. The use of an the embryo support during regeneration facilitates observation of the embryos placed thereon. The use of a support also promotes growth and facilitates the transfer of embryos to different media or treatments.
The pre-treatment maintains a large population of uniformly staged haploid microspores formed according to the method of the invention provides better targets for mutation, selection and transformation of plants. Using the inventive method, transgenic plants may be formed from microspores following pre-treatment by any conventional transformation method, such as bombardment with a transgene.
A pre-treatment to maintain microspores at a common stage of the cell cycle allows production of large number of embryos and regenerated plants from isolated microspores. The combination of cold plus sugar alcohol during the pre-treatment permits a much shorter treatment duration than using just cold treatment alone. Nearly all genotypes respond to this combined cold and sugar alcohol treatment.
The use of the combined cold plus sugar alcohol pre-treatment not only induces large numbers of embryoids but also tends to synchronize microspore cell divisions, thus leading to a more uniform microspore population. The likelihood of nuclear division during pre-treatment is reduced using the combined cold and sugar alcohol pre-treatment. This could be valuable for methodologies involving microspore transformation, testing expression of gene constructs, in vitro selection, or other microspore uses.
The use of arabinogalactan protein during induction of embryogenesis results in an increased yield of microspore embryos from pre-treated plant segments, thereby enhancing the efficiency of the method for embryogenesis.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.